The current quantitative methods of bilirubin have disadvantages such as high cost and low sensitivity. Due to the negative correlation between the level of serum bilirubin and the risk of cardiovascular diseases, a fluorescent ratiometric film sensor was developed aiming at bilirubin detection at low level concentration. Blue-emitting and red-emitting gold nanoclusters were assembled into the same film using layer-by-layer self-assembly technology. Detection of bilirubin was achieved based on the intensity ratio of the two nanoclusters. Bilirubin exposure causes fluorescent quenching of the film. The fluorescence intensity ratio of the two cluster probes had quantitative relationship versus bilirubin concentration. Based on this film sensor, a portable fluorescence detection system was designed for the ratiometric sensing of bilirubin. The hardware of the system was mainly composed of main control chip STM32F407, TSL237 and TSL238T optical frequency sensor. A light-avoiding dark room and detection light path were designed through three-dimensional printing to reduce the interference from ambient light and improve detection accuracy. Experimental results showed that the proposed detection system had strong anti-interference, good stability and accuracy. The linear coefficient of bilirubin detected by this system was 0.987. The system presented good results in reproducible experiments and possessed a good linear relationship with the data obtained by standard spectrofluorometer. The portable system is expected to detect serum bilirubin at low levels.